Streptomyces

Extracellular vesicle release is a wide-spread and broadly important phenomenon in bacteria. However, not much is known about the mechanism of vesicle release in Gram-positive bacteria. Observations of polarly growing Streptomyces venezuelae by live cell time-lapse imaging reveal release of extracellular membrane vesicles from tips of vegetative hyphae. Vesicle extrusion is associated with spontaneous growth arrests, but often the apical cell survives and can re-initiate growth by forming new hyphal branches. Treatment with vancomycin to block peptidoglycan synthesis leads to a high frequency of lysis and vesicle extrusion, where some hyphae can survive growth arrest and vesicle extrusion and reinitiate growth after antibiotic is washed away. The extruded vesicles do not contain nucleoids and do not appear able to proliferate. Vesicle extrusion is not affected by the Ser/Thr protein kinase AfsK that phosphorylates the DivIVA at hyphal tips, nor is it affected by the intermediate filament-like protein FilP that localizes in gradient-like structures at hyphal tips. Notably, hyphae of a scy mutant, which has an unstable apical polarisome structure, are prone to spontaneous growth arrests and vesicle extrusion even in the absence of antibiotic treatment, supporting the idea that the nature of the growth zone at the hyphal tips is important for this route of extracellular vesicle formation. We speculate that the propensity for vesicle extrusion is a direct consequence of how polar growth is organized at hyphal tips in Streptomyces , with the cell-wall sacculus being weak and susceptible to bursting at the apical zones of growth where peptidoglycan synthesis is primarily taking place.

Gram-positive Actinobacteria from the genus Streptomyces are best known for their morphological complexity and for their ability to produce numerous bioactive specialized metabolites with useful applications in human and veterinary medicine and in agriculture. In contrast, the ability to infect living plant tissues and to cause diseases of root and tuber crops such as potato common scab (CS) is a rare attribute among members of this genus. Research on the virulence mechanisms of plant-pathogenic Streptomyces spp. has revealed the importance of the thaxtomin phytotoxins as key pathogenicity determinants produced by several species. In addition, other phytotoxic specialized metabolites may contribute to the development or severity of disease caused by Streptomyces spp., along with the production of phytohormones and secreted proteins. A thorough understanding of the molecular mechanisms of plant pathogenicity will enable the development of better management procedures for controlling CS and other plant diseases caused by the Streptomyces .

The three nitrate reductases (Nar) of the saprophytic aerobic actinobacterium Streptomyces coelicolor A3(2) contribute to survival when oxygen becomes limiting. In the current study, we focused on synthesis of the Nar2 enzyme, which is the main Nar enzyme present and active in exponentially growing mycelium. Synthesis of Nar2 can, however, also be induced in spores after extended periods of anoxic incubation. The osdRK genes (oxygen stress and development) were recently identified to encode a two-component system important for expression of the nar2 operon in mycelium. OsdK is a predicted histidine kinase and we show here that an osdK mutant completely lacks Nar2 enzyme activity in mycelium. Recovery of Nar2 enzyme activity was achieved by re-introduction of the osdRK genes into the mutant on an integrative plasmid. In anoxically incubated spores, however, the osdK mutant retained the ability to synthesize NarG2, the catalytic subunit of Nar2. We could also demonstrate that synthesis of NarG2 in spores occurred only under hypoxic conditions; anoxia, as well as O2 concentrations significantly higher than 1 % in the gas-phase, failed to result in induction of NarG2 synthesis. Together, these findings indicate that, although Nar2 synthesis in both mycelium and spores is induced by oxygen limitation, different mechanisms control these processes and only Nar2 synthesis in mycelium is under the control of the OsdKR two-component system.

Actinomycete bacteria use polyprenol phosphate mannose as a lipid-linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. Strains of Streptomyces coelicolor with mutations in the gene ppm1, encoding polyprenol phosphate mannose synthase, and in pmt, encoding a protein O-mannosyltransferase, are resistant to phage ϕC31 and have greatly increased susceptibility to some antibiotics, including vancomycin. In this work, second-site suppressors of the vancomycin susceptibility were isolated. The suppressor strains fell into two groups. Group 1 strains had increased resistance to vancomycin, teicoplanin and β-lactams, and had mutations in the two-component sensor regulator system encoded by vanSR, leading to upegulation of the vanSRJKHAX cluster. Group 2 strains only had increased resistance to vancomycin and these mostly had mutations in sco2592 or sco2593, genes that are derepressed in the presence of phosphate and are likely to be required for the synthesis of a phosphate-containing extracellular polymer. In some suppressor strains the increased resistance was only observed in media with limited phosphate (mimicking the phenotype of wild-type S. coelicolor ), but two strains, DT3017_R21 (ppm1-vanR -) and DT3017_R15 (ppm1- sco2593 -), retained resistance on media with high phosphate content. These results support the view that vancomycin resistance in S. coelicolor is a trade-off between mechanisms that confer resistance and at least one that interferes with resistance mediated through the sco2594-sco2593-sco2592 operon.

Genes encoding dodecin proteins are present in almost 20 % of archaeal and in more than 50 % of bacterial genomes. Archaeal dodecins bind riboflavin (vitamin B2), are thought to play a role in flavin homeostasis and possibly also help to protect cells from radical or oxygenic stress. Bacterial dodecins were found to bind riboflavin-5′-phosphate (also called flavin mononucleotide or FMN) and coenzyme A, but their physiological function remained unknown. In this study, we set out to investigate the relevance of dodecins for flavin metabolism and oxidative stress management in the phylogenetically related bacteria Streptomyces coelicolor and Streptomyces davawensis. Additionally, we explored the role of dodecins with regard to resistance against the antibiotic roseoflavin, a riboflavin analogue produced by S. davawensis. Our results show that the dodecin of S. davawensis predominantly binds FMN and is neither involved in roseoflavin biosynthesis nor in roseoflavin resistance. In contrast to S. davawensis, growth of S. coelicolor was not reduced in the presence of plumbagin, a compound, which induces oxidative stress. Plumbagin treatment stimulated expression of the dodecin gene in S. davawensis but not in S. coelicolor. Deletion of the dodecin gene in S. davawensis generated a recombinant strain which, in contrast to the wild-type, was fully resistant to plumbagin. Subsequent metabolome analyses revealed that the S. davawensis dodecin deletion strain exhibited a very different stress response when compared to the wild-type indicating that dodecins broadly affect cellular physiology.

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1 - mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1 - strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.

Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of antibiotics. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XL-related anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 73 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and a phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ- or IP-form, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ- and IP-form biosynthetic pathways.

MtrAB is a highly conserved two-component system implicated in the regulation of cell division in the Actinobacteria. It coordinates DNA replication with cell division in the unicellular Mycobacterium tuberculosis and links antibiotic production to sporulation in the filamentous Streptomyces venezuelae. Chloramphenicol biosynthesis is directly regulated by MtrA in S. venezuelae and deletion of mtrB constitutively activates MtrA and results in constitutive over-production of chloramphenicol. Here we report that in Streptomyces coelicolor, MtrA binds to sites upstream of developmental genes and the genes encoding ActII-1, ActII-4 and RedZ, which are cluster-situated regulators of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red). Consistent with this, deletion of mtrB switches on the production of Act, Red and streptorubin B, a product of the Red pathway. Thus, we propose that MtrA is a key regulator that links antibiotic production to development and can be used to upregulate antibiotic production in distantly related streptomycetes.

Polyfructans are synthesized from sucrose by plants (mostly inulin) and by both Gram-negative and Gram-positive bacteria (mostly levan). In the phylum Actinobacteria only levan synthesis by Actinomyces species has been reported. We have identified a putative fructansucrase gene (hugO) in Streptomyces viridochromogenes DSM40736 (Tü494). HugO was heterologously expressed and biochemically characterized. HPSEC-MALLS and 2D-1H-13C nuclear magnetic resonance (NMR) spectroscopy analysis showed that the fructan polymer produced in vitro has an Molecular Weight of 2.5*107 Da and is an inulin that is mainly composed of (β2–1)-linked fructose units. This is the first report of a fructansucrase from Streptomyces and an inulosucrase from Actinobacteria. Database searches showed that fructansucrases clearly occur more widely in streptomycetes. Analysis of the active site of HugO and other actinobacterial Gram-positive fructansucrases revealed that their +1 substrate-binding sites are conserved, but are most similar to those in Gram-negative fructansucrases. HugO also resembles Gram-negative fructansucrases in not requiring calcium ions for activity. The origin and properties of HugO and other actinobacterial fructansucrases thus clearly differ from those of previously characterized Gram-positive fructansucrases.

To study the link between energy metabolism and secondary metabolism/morphological development in Streptomyces, knockout mutants were generated with regard to the subunits of the cytochrome oxidase supercomplex (CcO) in Streptomyces coelicolor A3(2). All mutants exhibited an identical phenotype: viable but defective in antibiotic production and cell differentiation when grown in both complex and minimal media. The growth yield of the CcO mutant was about half of that of the WT strain on glucose medium while both strains grew similarly on maltose medium. Intracellular ATP measurement demonstrated that the CcO mutant exhibited high intracellular ATP level. A similar elevation of intracellular ATP level was observed with regard to the WT strain cultured in the presence of BCDA, a copper-chelating agent. Reverse transcriptase PCR analysis demonstrated that the transcription of ATP synthase operon is upregulated in the CcO mutant. Addition of carbonylcyanide m-chlorophenylhydrazone, an inhibitor of ATP synthesis, promoted antibiotic production and aerial mycelia formation in the CcO mutant and BCDA-treated WT cells. We hypothesize that the deficiency of CcO causes accumulation of intracellular ATP, and that the high ATP level inhibits the onset of development in S. coelicolor.